Liver cirrhosis is a common clinical chronic liver disease, and is a diffuse lesion damage of liver resulting from one or more prolonged or repeated insults. Liver cirrhosis is a pathologic process of abnormal proliferation of hepatic fibrous-connective tissue when necrosis of liver cells and inflammation occur. Excess hepatic fibrosis causes the atrophy and cirrhosis of liver, which eventually leads to decompensated cirrhosis or hepatic failure and results in patient's death.
Early in 2002, the WHO's statistics showed that nearly 80,000 death cases were caused by liver cirrhosis every year worldwide. Liver cirrhosis has been recognized as one of the ten death leading diseases by 2010. Liver cirrhosis has become a global public health concern because of its high incidence and severity. The most common cause of liver cirrhosis is hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Approximately one-third of the world population (2,000 million) has been or is being infected by hepatitis B virus, and 350 million of them have suffered the lifelong infection. Moreover, the infectious rate of HBV in China has reached 10% of the whole Chinese population. Consequently, the incidence of liver cirrhosis caused by HBV or HCV infection has been increasing year by year in China. According to an incomplete statistics analysis, there are about 20 million patients with chronic hepatitis B in China, whereas nearly 250,000˜300,000 of them may develop into liver cirrhosis, and 50,000˜200,000 may develop into liver cancer. Meanwhile, up to 200,000 people die of liver cirrhosis every year in China. Taken together, it would greatly decrease the incidence of liver cirrhosis and liver cancer, thereby reducing the death rate, cutting down the medical expenses and improving the life quality of patients, if the susceptible population could be identified so that early diagnosis and intervention could be applied. It also provides a tool to better understand the pathogenesis of liver cirrhosis and to search for an early intervention protocol, and eventually to better control this complicated disease.
Serum Amyloid A1 (SAA1) is an acute phase reactant protein comprised of 104 amino acids with a molecular weight of about 12-14 kDa under natural conditions. The SAA1 gene is located at chromosome 11 in human beings. SAA1 have been reported by the early researches as an acute phase inflammatory protein because SAA1 can be produced by liver activated macrophages and fibroblasts in an acute inflammation, with the concentration increasing up to 100-1000 folds of the normal value (generally the normal value is 910±270 μL). In addition, SAA1 is an apolipoprotein and can substitute to Apolipoprotien A1 (apoA1) in HDL during the acute phase inflammation thereof regulating cholesterol metabolism. However, recently many researches have shown that SAA1 is not only an acute phase inflammatory protein, but also an opsonin with ininnate immunity. It can interact with proinflammatory cytokines such as IL-6, TNF-α to regulate innate and adaptive immunity. It has been reported that SAA1 played important roles in pathogenesis of developing chronic inflammatory diseases and autoimmune diseases including diabetes, coronary heart diseases and rheumatoid arthritis (RA), etc.
Doctor He et al. in Hong Kong University discovered that SAA1 is significantly increased in the liver of patients suffering from liver cirrhosis and liver cancer. It is known that, after HBV infection, patients can experience three steps: hepatitis, cirrhosis and cancer. Liver cirrhosis develops from the chronic inflammation and/or liver cell necrosis resulting from virus infection and the proliferation of hepatic fibroblasts. It has not been reported yet as to whether SAA1 is directly involved in the aforemetioned pathological changes. Nevertheless, the researches of Pasteur Institute showed that SAA1 is closely interrelated with hepatitis virus infection. In HBV X gene transgenic mouse, the expression of SAA1 was significantly suppressed, indicating that SAA1 may be involved in HBV infection or subsequent pathological changes such as cirrhosis, liver cancer. Therefore, the present invention sets forth that SAA1 is related to the liver cirrhosis caused from hepatitis B, and provides the novel uses of SAA1 in detecting or determining the pathogenesis of liver cirrhosis, diagnosis and interventions.
Gene polymorphism refers to the presence of two or more non-continuous variant genes or genotypes or alleles in one biological species, also called as genetic polymorphism. The Single Nucleotide Polymorphism (“SNP”) of genes refers to the variants of a single nucleotide base group in one gene sequence, including the deletion, insertion, and substitution of a single nucleotide group. Human gene polymorphism is involved in the pathogenesis of disease and related to its diagnosis and treatment. Gene polymorphism is vital not only in illustrating the susceptibility and resistance to diseases and toxicants, and various clinical symptoms, but also in predicting the responses and outcomes of medical treatments. Therefore, the study of gene polymorphism has become a hot field in medical sciences.
As distinct racial differences are present in gene polymorphism, different races may reflect big diversities. Therefore, it's important to carry out the studies on the gene polymorphism in relation to liver cirrhosis in the Chinese population as it is vulnerable to the disease. By studying the polymorphism of susceptible genes in liver cirrhosis thereof establishing a detection method, people who carry the susceptible genotypes can be identified. It will be beneficial to the individual targeted therapy of liver cirrhosis. The polymorphism analysis is also important in the outcome prediction of medical treatment of liver cirrhosis patients.
It's known that SAA1 has three alleles: SAA1α, β and γ, and further comprises six genotypes: α/α, α/β, α/γ, β/β, β/γ, and γ/γ (see Table 1). Studies have shown varied proportions of different SAA1 genotypes among the races. In recent years, the correlation between SAA1 genotypes and diseases has been receiving more and more attentions. Many studies have demonstrated that SAA1 genotypes are well associated with some diseases. For example, in the study on SAA1 genotypes of 321 Japanese, Yamade et al. have found that the distribution frequency of allele α (1.1), β (1.5), γ (1.3) among Japanese are 0.310, 0.347 and 0.330 respectively. Ishii et al. have found that the most common genotype for secondary amyloidosis is SAA1γ/γ (1.3/1.3) in 127 RA patients. The occurrence of amyloidosis is highly related to the gene frequency of SAA1γ. In the γ/γ homozygote, the concentration of SAA1 in serum and the ratio of SAA1 to CRP, also an acute phase reactant are higher than those in other genotypes. While among Caucasians, amyloidosis is in direct proportion to the frequency of SAA1α homozygote. In the study of hemorrhagic fever in the Mediterranean, literatures reported that the incidence of SAA1α is seven times higher than the other genotypes. While in the SNP study of SAA1, it is also reported that HDL-C levels of different genotypes varied greatly. Nevertheless, as far as we know, there is no report on the correlation between SAA1 genotypes and liver cirrhosis.
Restriction Fragment Length Polymorphism (“RFLP”) is the commonly used method in studying gene polymorphism. This method involves numerous steps and has poor reproducibility, and is not convenient for large-scaled SNP analysis. Therefore, it is important to develop a convenient, fast, accurate and economic SAA1 gene polymorphism detection method for diagnosis of diseases.
By designing SAA1 allele specific reverse primer at which the end of 3′-terminus is located at the SNP site, and is further modified with thiophosphorylation, the present application has developed real-time allele-specific PCR to genotype SAA1. The present invention analyzes SAA1 genotypes and studies the correlation between the SAA1 genotypes and liver cirrhosis. The study results of the present invention show that the distribution of SAA1 genotypes in Chinese population is greatly different from that in the other populations, and the SAA1β/β homozygote do well correlate with the liver cirrhosis, and thereby can be used as a biomarker in the diagnosis and/or prognosis of the liver cirrhosis.
The present invention disclosed for the first time that there exists a positive correlation between SAA1β/β homozygote and hepatitis related liver cirrhosis. The SAA1β/β homozygote, as a biomarker and a high-risk factor of liver cirrhosis, can be used in the diagnosis of liver cirrhosis and the prognosis of hepatitis B patients. Furthermore, the present invention has also evaluated the diagnostic criteria of the SAA1β/β homozygote developing to liver cirrhosis. After a careful screen of the SAA1 homozygote of clinic samples from hepatitis B patients, liver cirrhosis patients, and normal controls with the real-time allele-specific PCR technology reported here, the present invention disclosed for the first time that the significant positive correlation between the SAA1β/β homozygote and liver cirrhosis. The SAA1β/β homozygote is a risk factor of developing liver cirrhosis and is highly valued in the prognosis of liver cirrhosis. Meanwhile, the SAA1β/β homozygote, as a biomarker of liver cirrhosis, has a great significance for developing non-invasive diagnosis reagents for liver cirrhosis. Moreover, the designed primers of the present invention are suitable for detecting human SAA1 SNP with the real-time allele-specific PCR technology. The technology of the present invention can screen liver cirrhosis susceptible individuals in a convenient, accurate and fast manner. The technology is applicable for the study of other SAA1 SNP related diseases.
TABLE 1SAA1 alleles and their corresponding amino acidsamino acid mutationAmino acid site5257SAA1 alleleαValAlaβAlaValγAlaAlaSAA1 genotypeβ/βAla/AlaVal/ValNon-β/βα/αVal/ValAla/Alaα/βVal/AlaAla/Valα/γVal/AlaAla/Alaβ/γAla/AlaVal/Alaγ/γAla/AlaAla/Ala